Metformin promotes osteogenic differentiation and prevents hyperglycaemia-induced osteoporosis by suppressing PPARγ expression

Metformin can prevent hyperglycaemia-induced osteoporosis and decrease the bone fracture rate, but the mechanism has not been fully elucidated. To reveal the mechanism by which metformin affects hyperglycaemia-induced osteoporosis, we treat a mouse osteoporosis cell model with metformin and find that osteoblast mineralization increases and PPARγ expression decreases. Single-cell mRNA sequencing analysis show that PPARγ is highly expressed in the bone tissue of osteoporosis patients, which highlights the role of PPARγ in osteoporosis. Furthermore, we find that PPARγ is the effector through which metformin prevents osteoporosis. We further examine the mechanism of PPARγ regulation and reveal that metformin regulates PPARγ expression through the AMPK pathway and that PPARγ affects osteoblasts through the endoplasmic reticulum stress (ERS) pathway. Moreover, we verify the association between the effect of metformin on bone metabolism and the expression of PPARγ in high-fat diet-induced diabetic rats. Thus, we identify and functionally validate that metformin prevents hyperglycaemia-induced osteoporosis by regulating the AMPK-PPARγ-ERS axis.


Introduction
The number of people with diabetes mellitus (DM) has quadrupled in the past three decades worldwide and may grow to 693 million by 2045 [1,2]. The prevalence of osteoporosis was reported to be 23.1% in women and 11.7% in men worldwide [3]. However, the prevalence rate of osteoporosis in type 2 diabetes mellitus (T2DM) patients is 32.1% in women and 21.2% in men [4]. In addition, bone fragility is a typical complication of diabetes, and the risk of fragility fractures is increased in diabetes patients [5]. Thus, diabetes is a risk factor for osteoporosis and fragility fractures.
Metformin is a widely used medication for the management of diabetes and related areas of clinical treatment and appears to function via multiple pathways [6][7][8]. Multinomial clinical studies have shown that metformin increases bone mineralization, reducing fracture risk in diabetes patients [9]. Cell line experiments also revealed that metformin improves bone formation by osteoblasts [10]. However, the molecular mechanism by which metformin protects osteoblasts has not been fully elucidated.
Peroxisome proliferator-activated receptor γ (PPARγ) is a ligandinducible transcription factor that is a member of the nuclear receptor superfamily and plays a key role in the regulation of cell differentiation and lipid metabolism [11]. Adenosine 5′-monophosphate-activated protein kinase (AMPK) activation and an increase in BMP2 are negatively correlated with the expression of PPARγ [12,13]. PPARγ correlates with insulin resistance and is considered to be a drug target for treating T2DM [14]. In addition, PPARγ activation can affect the structure of osteoblasts [15] and result in the loss of bone mass in the context of diabetes [16]. Thus, targeting PPARγ may be a promising approach to prevent osteoporosis. However, the regulation of PPARγ activity and the downstream effects of PPARγ on hyperglycaemia-induced osteoporosis are still unclear.
Hyperglycaemia can lead to metabolic disturbances via multiple pathways, such as the activation of Toll-like receptors (TLRs), inflammasome activation and endoplasmic reticulum stress (ERS) [17]. TLRs regulate osteoclast genesis and bone resorption through myeloid differentiation and β-interferon pathways [18]. The NLRP3 inflammasome plays a key role in the pathogenesis of osteoporosis by affecting the differentiation of osteoblasts and osteoclasts [19]. ERS induces osteoblast apoptosis and is related to metabolic bone disease [20]. In β-cells, hyperglycaemia-induced ERS is inhibited by metformin treatment [21] and PPARγ activation [22]. In human bronchial epithelial cells, AMPK alleviates ERS by inducing the ER chaperone ORP150 [23], and AMPK is closely related to the expression of PPARγ. However, the interplay among metformin, ERS and PPARγ in osteoblasts is still unknown.
In this study, to examine the regulatory relationship between metformin and the AMPK-PPARγ-ERS axis in osteoporosis prevention, we treated mouse osteoblasts and a diabetic rat model with metformin and then explored the mechanism by which metformin affects hyperglycaemia-induced osteoporosis.
For high glucose intervention, MC3T3-E1 cells were cultured in high glucose medium (base medium supplemented with 25 mM glucose) for 14 days. For metformin intervention, MC3T3-E1 cells were cultured in medium containing different concentrations of metformin (25,50, 100 μM metformin in base medium) for 14 days. For thapsigargin intervention, MC3T3-E1 cells were cultured in base medium containing 100 nM β-thapsigargin (Beyotime) for 1 day.

Analysis of PPARγ protein expression levels in human tissue
PPARγ expression in human bone tissue cells was analysed using published single-cell mRNA sequencing datasets [26,27] after removing the bulk gene background and filtering out cells with fewer than 500 detected transcripts. The matrix of digital gene expression data was transformed with ln(CPM/100+1), and downstream procedures for filtering and reducing dimensionality were performed using Seurat version 4.0.5. Cell type annotation was performed using scMRMA version 1.0. All genes were used for initial principal component analysis, and the first 10 principal components were used for nonlinear dimensionality reduction (t-distributed stochastic neighbourhood embedding, tSNE) analysis.
PPARγ expression in human bone tissue from osteoporotic and healthy individuals was analysed by western blot analysis. A total of 20 osteoporosis patients and 10 healthy controls with T2DM were recruited for this experiment. Bone samples were obtained from patients with fractures during surgery. This study was approved by the Ethics Committee of the First Affiliated Hospital of Fujian Medical University (Fuzhou, China; NO. IEC-FOM-013-2.0).

Animal experiments
Five-to six-week-old male Sprague-Dawley rats were used. After a 1-week acclimation period, the animals were weighed, measured, and divided into two groups: a regular control diet (NC, n=20) and a high-fat diet (HFD, n=20) group. HFD rats received an HFD for 16 weeks to induce obesity. To induce type 2 diabetes, HFD rats were administered with 30 mg/kg body weight streptozotocin (Sigma Aldrich, St Louis, USA) by injection. Regular control diet rats were injected with an equivalent volume of saline. After the onset of diabetes, a total of 20 model rats were grouped into the metformintreated group (900 mg/kg/day for 20 weeks, n=10) or the T2DM group (gastric perfusion of an equal volume of saline, n=10) using a random number table. Body weight and length were recorded once per week for the duration of the study. All rat experiments were approved by the Fujian Medical University Institutional Animal Care and Use Committee.

Analysis of bone mineral density and bone mineral levels
Bone mineral density was analysed as previously described [28,29]. Briefly, the rats were anaesthetized by an intraperitoneal injection of 10% chloral hydrate (0.03 mL/kg), and bone mineral density and bone mineral levels were measured by dual energy X-ray absorptiometry with a DEXA scanner (GE, Milwaukee, USA).

ELISA
To analyse serum bone turnover markers, osteocalcin, ALP and TRAP levels in abdominal aorta blood samples were measured. Briefly, after the cells were treated as indicated, the supernatant was collected in a sterile tube. The supernatant was centrifuged at 2850 g

Western blot analysis
Control and experimental rats were fasted overnight for 8 h and then anaesthetized by an intraperitoneal injection of 10% chloral hydrate (0.03 mL/kg). Femur tissue was collected, and the muscle and connective tissue were removed, rinsed with saline and stored in liquid nitrogen before analysis. The femur was removed from liquid nitrogen and quickly crushed, and then the bone fragments were ground into powder with liquid nitrogen. The powder was placed in a sterile tube. Then, 1 mL of protein lysate was used per 100 mg of bone tissue, placed on ice for 30 min, shaken once every 10 min, and centrifuged at 12,000 g for 10 min at 4°C. The supernatant was stored at -80°C. Protein concentrations in femur and MC3T3-E1 cells were determined by using bicinchoninic acid (BCA) protein assay kit (Sigma-Aldrich).

Microcomputed tomography (micro-CT)
The first to fourth lumbar vertebrae were collected from the rats and placed in 4% paraformaldehyde for 24 h. The samples were scanned by micro-CT (Scanco Medica, Zurich, Switzerland). The scanning conditions were as follows: 70 kV voltage, 200 μA current, and 9 μm resolution. After the samples were scanned, the images were reconstructed using Mimics 19.0. DataViewer was used to adjust the images.

Statistical analysis
Statistical analysis was performed using one-way ANOVA. Bivariate correlation analysis was used to analyse the correlation between the levels of pAMPK and the effect of metformin in glucotoxicity osteoblasts. Data are expressed as the mean±SEM. P values<0.05 were considered statistically significant.

Metformin improves osteoblast differentiation and suppresses PPARγ expression in osteoblasts exposed to glucotoxicity
To examine the effect of metformin on osteoblasts, we treated the streptozotocin-induced osteoporosis cell model with different concentrations of metformin (25,50, and 100 μM). The results showed that 50 and 100 μM metformin significantly increased mineralization ( Figure 1A) in hyperglycaemic osteoblasts. In the 100 μM metformin treatment group, the protein level of PPARγ was significantly decreased, and the levels of p-AMPK and BMP-2 were increased ( Figure 1B). These findings indicate that metformin improves osteoblast differentiation and suppresses PPARγ expression in osteoblasts.

PPARγ is highly expressed in the bone tissue of osteoporosis patients
To confirm that PPARγ is the specific effector molecule of osteoporosis, we analysed the expression level of PPARγ in human bone tissue using published single-cell mRNA sequencing datasets [26,27]. We found that PPARγ was highly expressed in preosteoblasts (1970/5729 cells) but was rarely expressed in mature osteoblasts (201/1528 cells) (Figure 2A-C) and was not expressed in other cells, such as chondrocytes, neutrophils and endothelial cells ( Figure 2D-E). In addition, we analysed the PPARγ protein levels in the bone tissue of 30 T2DM patients, including 20 osteoporosis patients and 10 healthy individuals ( Figure 2F). The results showed that the PPARγ protein levels in osteoporosis patients were significantly higher than those in healthy controls. Thus, these results suggest that PPARγ is highly expressed in the bone tissue of osteoporosis patients and may play an important role in osteogenic differentiation.

PPARγ plays an important role in metformin-mediated effect on hyperglycaemic osteoblasts
To verify that metformin improves mineralization by affecting PPARγ, we altered the expression or activity of PPARγ in a metformin treatment assay. In this assay, we altered PPARγ levels through overexpression and RNAi-mediated knockdown ( Figure  3A). The results showed that downregulating PPARγ levels improved the metformin-mediated effect on hyperglycaemic osteoblasts; osteoblast viability ( Figure 3B) and mineralization ( Figure  3C), the secretion of ALP ( Figure 3D) and OCN ( Figure 3E), and the expression of BMP2 ( Figure 3F) were increased. Conversely, the increased PPARγ level in osteoblasts weakened the metforminmediated effect on hyperglycaemic osteoblasts ( Figure 3B-F).
Furthermore, we explored whether PPARγ activation can alter the effect of metformin on hyperglycaemia-induced osteoporosis. We used a PPARγ inhibitor (GW9662) and agonist (PIO) to selectively block or activate PPARγ activity, respectively. It was found that inhibiting PPARγ activity increased the effects of metformin, increasing osteoblast viability ( Figure 3G), mineralization ( Figure  3H), the secretion of ALP ( Figure 3I) and OCN (Figure 3J), and the expression of BMP2 ( Figure 3K). In addition, activating PPARγ decreased the effects of metformin ( Figure 3G-K). These findings indicate that PPARγ plays a key role in the protective effect of metformin on hyperglycaemic osteoblasts.

PPARγ mediates the effects of metformin through the AMPK pathway
We examined whether PPARγ affects the activity of metformin through the AMPK pathway. Dorsomorphin (compound C) is an AMPK inhibitor. After inhibiting the activity of AMPK, we found that the protective effect of metformin was reduced: compared with those in the HG+MF group, osteoblast viability ( Figure 4A) and mineralization ( Figure 4B), the secretion of ALP ( Figure 4C) and OCN ( Figure 4D), and the expression of BMP2 ( Figure 4E) in the HG+MF+Compound C group were decreased. Compound C also weakened the inhibitory effect of metformin on the expressions of pAMPK, PPARγ, PERK, ATF4, and CHOP ( Figure 4E,F) in hyperglycaemic osteoblasts. These findings suggest that PPARγ affects the activity of metformin through the AMPK pathway.

PPARγ affects hyperglycaemia-induced osteoporosis through the ERS pathway
ERS is an important inducer of osteoporosis [20], and metformin inhibits ERS in adipose tissue [21]. Here, we examined the role of

Metformin prevents osteoporosis by suppressing PPARγ 397
PPARγ in ERS after metformin treatment. The results showed that metformin significantly reduced the expression of ERS-associated proteins, such as PERK, ATF4, and CHOP ( Figure 5A), in hyperglycaemic osteoblasts; downregulating PPARγ levels enhanced the effect of metformin on PERK, CHOP and ATF4 in the context of hyperglycemia ( Figure 5B). We also induced PPARγ overexpression in osteoblasts and found that PPARγ overexpression reduced the antagonistic effect of metformin on aberrant gene expression induced by hyperglycaemia in osteoblasts ( Figure 5B). β-thapsigargin is an activator of ERS and was used to examine whether PPARγ is involved in metformin-mediated alleviation of ERS. Metformin inhibited the increased protein levels of PERK, CHOP and ATF4 induced by β-thapsigargin ( Figure 5C). Silencing of PPARγ enhanced the regulatory effect of metformin on the protein levels of PERK, CHOP and ATF4 ( Figure 5D). The protective effect of metformin was weakened by PPARγ overexpression ( Figure 5D). These results suggest that PPARγ is involved in the effects of metformin by alleviating hyperglycaemia-induced ERS.

PPARγ levels correlate with bone metabolism in metformin treated rats
To examine the protective effect of metformin against hyperglycemia-induced osteoporosis, the bone metabolism of diabetic rats was examined after treatment with metformin. Diabetes was induced in rats by a high-fat diet and streptozotocin. The results showed that metformin reduced fasting blood glucose ( Figure 6A) and increased insulin secretion (Figure 6B), the abundance of thin tissue ( Figure  6D), the fat mass in the torso (Figure 6E), and BMD in the whole body ( Figure 6F). However, no significant difference was observed in the weight ( Figure 6C) or bone mineral levels ( Figure 6G) between the DM and DM+MF groups. The micro-CT results showed that metformin treatment promoted bone formation in torso tissue ( Figure 6H). Bone metabolism markers were examined, and metformin treatment increased the levels of ALP ( Figure 6I) and

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Metformin prevents osteoporosis by suppressing PPARγ OCN ( Figure 6J) and decreased the secretion of TRAP5b ( Figure  6K). These results demonstrate that metformin improves hyperglycaemia-induced osteoporosis. Furthermore, we analysed the PPARγ pathway in the DM and DM+MF groups. The results showed that metformin treatment increased the expressions of BMP2 and pAMPK ( Figure 6L) and reduced the expressions of PPARγ ( Figure 6L), PERK, ATF4, and CHOP ( Figure 6M) in femur tissues. These findings indicate that metformin improves bone metabolism in diabetic rats, which correlates with the PPARγ-related pathway.

Discussion
In this study, we demonstrated that metformin enhanced osteoblast differentiation and downregulated the protein level of PPARγ in an osteoporosis cell model. Increasing the activity of PPARγ weakened these effects of metformin, whereas inhibiting the activity of PPARγ had the opposite effect. Additionally, we found that metformin regulated PPARγ function through the AMPK pathway. PPARγ mediates the effect of metformin via the ERS pathway. Furthermore, we found a relationship between the effect of metformin on bone metabolism and the activity of PPARγ in diabetic rats.

Metformin prevents osteoporosis by suppressing PPARγ 399
Previous experiments confirmed that the pathway by which metformin protects against hyperglycaemia in osteoblasts is related to a reduction in PPARγ [30]. However, what is the association between metformin and PPARγ? There is no related literature available. It has been verified that metformin protects against hyperglycaemia by regulating AMPK signalling [31]. Furthermore, our results showed that AMPK activity is negatively correlated with PPARγ expression level. Therefore, we hypothesized that metfor-min-mediated regulation of PPARγ expression might be related to AMPK. Thus, we inhibited AMPK and then observed whether the effect of metformin could be diminished in hyperglycaemic osteoblasts and whether metformin still could affect the expression of PPARγ. Our results revealed that the effect of metformin in this context was, to a certain extent, AMPK dependent.
During diabetes, bone strength is decreased, and bone fragility is increased, which is induced by high glucose toxicity, leading to the

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Metformin prevents osteoporosis by suppressing PPARγ occurrence and development of diabetic osteoporosis [32]. Our results indicate that metformin decreases the expressions of ERSrelated proteins in hyperglycaemic osteoblasts. Current research also suggests that metformin has a protective effect on hyperglycaemic osteoblasts [33]. We confirmed that metformin could inhibit PPARγ expression and thus attenuate glucotoxicity in osteoblasts. Next, we examined the ways by which metformin mediates its effects. Previous research has shown that ERS is a central mechanism of injury induced by high glucose in osteoblasts [15]. Studies from other laboratories have also demonstrated that metformin can prevent renal fibrosis by relieving ERS [34]. Our study verified the efficacy of metformin on ERS in osteoblasts.
In summary, we show that metformin enhances osteoblast differentiation by regulating PPARγ expression. Furthermore, the AMPK and ERS pathways are upstream and downstream of PPARγ, respectively. We conclude that: (i) PPARγ is the effector through which metformin prevents osteoporosis. (ii) PPARγ levels are related to osteoporosis in diabetes patients. (iii) Metformin prevents hyperglycaemia-induced osteoporosis by regulating the AMPK-PPARγ-ERS axis. Our findings will expand treatment strategies for hyperglycaemia-induced osteoporosis and may also expand metformin treatment to other PPARγ-related diseases.